processes Article Methanogens Diversity during Anaerobic Sewage Sludge Stabilization and the Effect of Temperature Tomáš Vítˇez 1,2, David Novák 3, Jan Lochman 3,* and Monika Vítˇezová 1,* 1 Department of Experimental Biology, Faculty of Science, Masaryk University, 62500 Brno, Czech Republic; [email protected] 2 Department of Agricultural, Food and Environmental Engineering, Faculty of AgriSciences, Mendel University, 61300 Brno, Czech Republic 3 Department of Biochemistry, Faculty of Science, Masaryk University, 62500 Brno, Czech Republic; [email protected] * Correspondence: [email protected] (J.L.); [email protected] (M.V.); Tel.: +420-549-495-602 (J.L.); Tel.: +420-549-497-177 (M.V.) Received: 29 June 2020; Accepted: 10 July 2020; Published: 12 July 2020 Abstract: Anaerobic sludge stabilization is a commonly used technology. Most fermenters are operated at a mesophilic temperature regime. Modern trends in waste management aim to minimize waste generation. One of the strategies can be achieved by anaerobically stabilizing the sludge by raising the temperature. Higher temperatures will allow faster decomposition of organic matter, shortening the retention time, and increasing biogas production. This work is focused on the description of changes in the community of methanogenic microorganisms at different temperatures during the sludge stabilization. At higher temperatures, biogas contained a higher percentage of methane, however, there was an undesirable accumulation of ammonia in the fermenter. Representatives of the hydrogenotrophic genus Methanoliea were described at all temperatures tested. At temperatures up to 50 ◦C, a significant proportion of methanogens were also formed by acetoclastic representatives of Methanosaeta sp. and acetoclastic representatives of the order Methanosarcinales. The composition of methanogens in the fermenter significantly changed at 60 ◦C when typically thermophilic species, like Methanothermobacter marburgensis, appeared. A decrease in the diversity of methanogens was observed, and typical hydrogenotrophic methanogenic archaea isolated from fermenters of biogas plants and anaerobic wastewater treatment plants represented by genus Methanoculleus were no longer present. Keywords: anaerobic stabilization; sewage sludge; temperature; methanogenic archaea 1. Introduction Wastewater treatment is an important process ensuring sustainable development of human society. On average, 144 L of water per person per day is supplied by households in Europe [1]. Most of this water, along with the water from the industry, will become wastewater after use. This water is discharged to wastewater treatment plants (WWTPs). In the process of wastewater treatment to purified water, we also get other products that we need to handle, as gravel, sand, screenings and sewage sludge. Gravel and sand are usually landfilled, incinerated, used for reclamation or construction purposes. Screenings are generally landfilled or incinerated [2], and sewage sludge remains as a product for wastewater treatment. Typical sewage sludge production in wastewater treatment varies from 35 to 85 g dry solids per population equivalent per day [3]. Sewage sludge should not be perceived as waste with problematic uses but as a valuable and well-utilized source of nutrients and energy. In recent years, sewage sludge has been mostly applied to the agricultural land as a fertilizer as it contains about Processes 2020, 8, 822; doi:10.3390/pr8070822 www.mdpi.com/journal/processes Processes 2020, 8, 822 2 of 12 5% of nitrogen, phosphorus, and iron and 2% of potassium [4]. Today, however, due to the high content of heavy metals and the presence of xenobiotic compounds, the use of sludge is being abandoned, and other possible alternatives are being sought [5]. Incineration of sludge is economically demanding due to the need to reduce the water content. Gasification, pyrolysis, combustion, and wet oxidation are used for the energy utilization of sewage sludge [4]. The disadvantage is the large quantity of flue gas and ash formed during sewage sludge incineration and the emissions of dioxins, furans, mercury, and other heavy metals, NOx,N2O, and CO [4]. Anaerobic sewage sludge stabilization is a method by which energetically utilizable biogas while reducing sludge mass is produced. An essential parameter for sludge stabilization is the temperature and hydraulic retention time (HRT) at which the stabilization process takes place [6]. The usual HRT for anaerobic sewage sludge stabilization ranges from 18 to 30 days. Some wastewater treatment plants undergo anaerobic sludge stabilization at mesophilic conditions. The advantage is the stability of the whole process, low costs of operating the reactors, and better sludge dewatering. The disadvantage is a longer HRT and lower biogas production. The thermophilic process produces higher amounts of biogas, possibly a higher percentage of methane in biogas, lower levels of pathogenic microorganisms in the sludge and shorter HRTs. The disadvantage is then worse drainability and worse process stability. These facts are described in many literary sources [7–11]. Also, lower methanogenic diversity in plants, operating at thermophilic temperatures was reported [12,13]. However, it is necessary to understand the essence of the whole process, the interplay of different groups of microorganisms, whose final product of metabolism is methane. Beside the fact, that temperature has important impact on whole microbial diversity in fermenters, in this work, we were interested, how the composition of methanogenic community’s changes depending on the full range of temperatures at which biogas transformation takes place and how the temperature range from 20 ◦C to 60 ◦C affects the production and quality of the biogas produced. The diversity of specific methanogens at such a wide temperature range of this anaerobic process has not yet been published in the literature. The detailed knowledge of the involvement of different methanogen groups, and their temperature requirements associated with maximum methane production, will enable the optimization of the input parameters in the anaerobic sewage sludge stabilization process at each wastewater treatment plant. 2. Materials and Methods 2.1. Analytical Methods Dry matter (DM) content was determined by drying at 105 C 5 C to constant weight followed ◦ ± ◦ by cooling in a desiccator. Organic dry matter (oDM) content was determined by incineration of the samples in a muffle furnace at 550 C 5 C using a furnace LMH 11/12 (LAC Ltd., Židlochovice, ◦ ± ◦ Czech Republic). For pH, redox, conductivity determination multi-parameter instrument WTW 3410 (Xylem Analytics Germany Sales GmbH & Co. KG, WTW, Weilheim, Germany) was used. For fatty acids and ammonia determination, ITP/CZE IONOSEP 2005 Analyzer (RECMAN Ltd., Ostrava, Czech Republic) was used. 2.2. Biomethanation Assay Biomethanation assay was performed in accordance with standard VDI 4613. Eight fermenters with the volume of 120 L each, were used (Figure1). Processes 2020, 8, 822 3 of 12 Processes 2020, 8, x 3 of 12 FigureFigure 1.1. Lab view view and and fermenter fermenter scheme, scheme, (1) (1) hot-water hot-water he heatingating system, system, (2) (2) electric electric heating heating coil, coil, (3) (3)electric-driven electric-driven agitator agitator with with a afrequency frequency converte converter,r, (4) (4) two two temperature temperature sensors forfor hot-waterhot-water andand fermenterfermenter content,content, (5)(5) gasgas meter,meter, (6)(6) samplingsampling pointpoint forfor biogasbiogas analysis,analysis, (7)(7) samplingsampling pointpoint forfor thethe fermentedfermented substrate substrate and and (8) (8) computer computer control control system. system. InIn thethe first first day day (day (day 0) of0) experiments,of experiments, all fermenters all fermenters were were filled upfilled with up 50 with L of 50 inoculum L of inoculum (sludge from(sludge anaerobic from anaerobic stabilization) stabilization) and 50 L and of primary 50 L of sludge,primary as sludge, a source as ofa source nutrients. of nutrients. Sludge samples Sludge weresamples collected were atcollected wastewater at wastewat treatmenter treatment plant 513,000 plant PE, 513,000 Modˇrice,Czech PE, Modřice, Republic. Czech Republic. WWTP Modˇrice WWTP anaerobicModřice sewageanaerobic sludge sewage stabilization sludge isstabilization operated at mesophilicis operated temperature at mesophilic (34 ◦ C).temperature Anaerobic (34 sewage °C). sludgeAnaerobic mixture sewage characteristics sludge mixture were characteristics pH = 7.2, dry matterwere pH (DM) = 7.2, content dry matter 2.64% (DM) and organic content dry 2.64% matter and (oDM)organic content dry matter 56.90%. (oDM) Before content the biomethanation 56.90%. Before assay the biomethanation starts, the fermenters assay werestarts, tempered the fermenters to the desiredwere tempered temperature. to the Fermenters desired temper No. 1,ature. 2 (60 FermentersC 0.5 C), No. fermenters 1, 2 (60 No.°C ± 3, 0.5 4 (50°C), fermenters0.5 C), fermenters No. 3, 4 ◦ ± ◦ ± ◦ No.(50 ± 5, 0.5 6 (40 °C), 0.5fermentersC), fermenter No. 5, No.6 (40 7 ± and 0.5 8°C), (20 fermenter0.5 C). TheNo. retention7 and 8 (20 time ± 0.5 (RT) °C). was The 21 retention days, and time the ± ◦ ± ◦ low-speed(RT) was 21 agitator days, and was the operated low-speed at 60 agitator rpm with was 1 minute operated agitation at 60 rpm time with alternated 1 minute with agitation 60 minutes time ofalternated resting time. with During60 minutes